The authors would like to thank Richard Rest for the generous gift of anthrolysin O, Michael Caparon for the generous gift of the SLO plasmid and Jonathon Franks for technical assistance. This work was funded by NIH grants T32CA82084 (PAK), and R01AI072083 (RDS).

1. Purification of Streptolysin O (SLO)
<ol>
	<li>Inoculate 20 ml overnight culture of BL21 GOLD cells containing pBADgIII-SLOhis plasmid9 into 0.5 L LB broth and add 500 &mu;l 50 mg/ml ampicillin. Shake culture at 225 rpm at 37 &deg; C until OD600 = 0.6, usually ~1.5 hr. Centrifuge 1 ml bacteria (considered T0) at 10,000 x g 5 min, dissolve pellet in 140 &mu;l 1x SDS sample buffer/1 OD and sonicate to shear DNA for protein purity analysis.</li>
	<li>Induce bacteria with the addition of 5 ml 20% arabinose to culture, shake at 225 rpm at room temp for 3 hr. Collect 1 ml of bacteria, centrifuge and dissolve in 1x SDS sample buffer as above for T3 time point for purity analysis.</li>
	<li>Collect remaining bacteria in 500 ml centrifuge bottle, spin 12,000 x g for 12 min at 4 &deg;C (8,500 rpm Sorvall GS-3 rotor). Decant the supernatant, store pellet at -80 &deg;C overnight or longer if needed.</li>
	<li>Resuspend the frozen pellet on ice in 10 ml Lyse/Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) supplemented with 400 &mu;l 25% Triton X-100, 100 &mu;l 100 mg/ml lysozyme, 50 &mu;l 200 mM phenylmethylsulfonyl fluoride (PMSF). Transfer the resuspended pellet to 50 ml Oak Ridge tube.</li>
	<li>Sonicate lysate 5 times for 30 sec on ice with 30 sec intervals. Spin sonicated lysate in oak ridge tube at 39,000 x g for 20 min at 4 &deg;C (18,000 rpm Sorvall SS-34 rotor).</li>
	<li>While the lysate is spinning, wash 1 ml Ni-NTA agarose with 10 ml Lyse/Wash buffer and spin at 1,200 rpm for 5 min at 4 &deg;C. (all further washes/elutions use centrifugation under these conditions). Aspirate wash.</li>
	<li>Add bacterial supernatant from Step 1.5 to Ni-NTA agarose, shake gently for 2.5 hr at 4 &deg;C. Spin to collect supernatant. Combine 30 &mu;l of supernatant with 10 &mu; 4x SDS sample buffer and save for purity analysis. Wash the beads 4 times in Lyse/Wash buffer and once in Tris/salt buffer (50 mM Tris, 300 mM NaCl, pH 8.0).</li>
	<li>Elute 3 times by adding 1 ml Elution buffer (250 mM imidazole in Tris/salt buffer) and 5 &mu;l 1 M dithiothreitol (DTT) to beads, incubating on ice for 10 min, spinning and collecting the supernatant. SLO is a very redox-sensitive protein, and must be kept on ice at all times. Once reduced, the protein will rapidly lose its activity if warmed to 37 &deg;C, even for a short period of time.</li>
	<li>Wash 500 &mu;l polymixin-conjugated agarose in elution buffer, spin at 10,000 x g for 1 min at 4 &deg;C and resuspend in 500 &mu;l. To deplete endotoxin, add 200 &mu;l beads to each elution and shake 4 &deg;C for 30 min. Spin at 10,000 x g for 1 min at 4 &deg;C, and save the supernatant. Combine 30 &mu;l of each elution with 10 &mu;l 4x SDS sample buffer for SDS-PAGE analysis.</li>
	<li>Test the purity of elutions by SDS-PAGE. Boil samples saved for gel analysis at 95 &deg;C for 5 min and load 10 &mu;l on a 10% gel. Stain gel with Coomassie to visualize protein (Figure 2A). SLO is 69 kDa. Perform a Bradford Assay to determine protein concentration (typical yield is 4 mg/ml for first two elutions, but may vary by bacterial strain and plasmid used).</li>
	<li>Test the hemolytic activity of each elution (see below). Pool elutions where purity, concentration and hemolytic activity are satisfactory. Aliquot SLO in single-use aliquots (typically 5-10 &mu;l), freeze on dry ice and store at -80 &deg;C.</li>
</ol>
2. Hemolytic Assay
<ol>
	<li>Wash sheep red blood cells (RBCs) in RBC Assay buffer (0.3% bovine serum albumin (BSA), 2 mM CaCl2, 10 mM Hepes, pH 7.4), spin at 1,200 rpm for 5 min, and resuspend at 2.5% RBCs in 10 ml RBC Assay buffer.</li>
	<li>Add 10 &mu;l RBC assay buffer/well in 96-well V-bottom plate on ice. Serially dilute each elution in duplicate. Typical dilution ranges are 1:1,000 to 1:512,000. Include 3 wells with no toxin and 3 wells with 10 &mu;l 2% Triton X-100 as minimum and maximum wells, respectively.</li>
	<li>Cover plate and incubate at 37 &deg;C 30 min. Centrifuge at 1,200 rpm for 5 min. Transfer 70 &mu;l of supernatant to a flat bottom 96 well plate. Read A405. One unit is the dilution of toxin required for 50% lysis of the RBCs. Toxin activity is 1 unit* dilution factor/0.01 ml.</li>
</ol>
3. Cell Lytic Assay
<ol>
	<li>Harvest cells, count, spin. Resuspend at 2x106/ml in buffer R/B (RPMI with 2 mM CaCl2, 0.5% BSA) and 20 &mu;g/ml propidium iodide. Add 100 &mu;l cells to a 96-well V-bottom plate.</li>
	<li>Serially dilute SLO to 2x final concentration in buffer R/B, add 100 &mu;l toxin or 100 &mu;l buffer R/B to cells. Incubate 5 min 37 &deg;C. Typical final concentrations range from 2,000 U/ml to 31.25 U/ml.</li>
	<li>Run cells on flow cytometer and collect data with filters for phycoerythrin (PE). A one-log shift represents transiently permeabilized cells while a 3 log shift indicates dead cells4. Calculate the specific lysis of the cells by subtracting the percentage of dead cells in the control (%PIhighctl) from the experimental (%PIhighexp), as follows: specific lysis= (%PIhighexp-%PIhighctl)/(100-%PIhighctl)*100</li>
</ol>
4. Micropipette Delivery of Toxin to Cells in Culture Dishes
<ol>
	<li>One day prior to experiment, plate 2x105 macrophages on a collagen-coated glass-bottom 35 mm dish.</li>
	<li>Turn on microscope and microinjector. Allow heated stage time to warm to 37 &deg;C. The choice of microscope usually depends on what is locally available. The microscope needs an inverted stage, a stage capable of heating the dishes to 37 &deg;C, excitation/emission filter cubes appropriate for the dye chosen, and the space to physically connect the microinjector. A Bertrand lens is helpful for microinjection, but not required. The computer driving the microscope needs sufficient memory to collect and store data.</li>
	<li>Label cells for 30 min with dye at 37 &deg;C. Depending on the assay, labeling may be done with 5 &mu;l Fura2 AM in 1 ml PBS or 2 &mu;l calcein AM in 1 ml full media. For Fura2, excitation/emission is collected for 340/510 nm and 380/510 nm, and the ratio of 340/380 signals determines calcium flux. For calcein, excitation/emission is 495/515 nm while ethidium homodimer is 525/620 nm and APC is 650/660 nm. Other labels may be chosen as well.</li>
	<li>Wash cells with PBS and place in 1 ml RPMI supplemented with 2 mM CaCl2. Mount on microscope.</li>
	<li>Dilute toxin and dextran in water, and centrifuge at 20,000 x g 10 min for 4 &deg;C. Typically, 1 &mu;l SLO and 4 &mu;l 10 mg/ml dextran-555 is diluted in 6 &mu;l water. Dextran or another fluorescent fluid-phase molecule is used to verify that the femto-tip is not clogged, and injects as desired.</li>
	<li>Load femto-tip from rear with 0.07 &mu;l of diluted toxin using a microloader.</li>
	<li>Load femto-tip onto microinjector. Adjust angle of injector so that the tip will sit over the center of the cells with room to move in all directions. Clear z-limit. Injection settings should be injecting for 0.5 sec at 120 psi with 20 psi back pressure. Lower tip until it enters the medium.</li>
	<li>Using the Bertrand lens, center the tip and follow the tip as it is lowered closer to the cells. Once you lose focus, switch back to normal optics. The needle shadow should be apparent in the field. Focus above the cells and lower the needle until it is in focus. Focus back on the cells and carefully bring the needle adjacent to a cell. Set the z-limit for injection. Commence imaging, move tip to desired position, inject to release toxin at the desired time point. Raise the needle, move to a new region of cells and inject. Move needle to home position to prevent any undesired toxin leakage from the needle.</li>
</ol>

<ol>
	<li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 3185-3190 (2001).</li><li>Walev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+regulates+IL-1+beta+processing+via+calcium-independent+phospholipase+A2.">Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2.</a> J. Immunol. 164, 5120-5124 (2000).</li><li>Scolding, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+removal+by+oligodendrocytes+of+membrane+attack+complexes+formed+by+activated+complement.">Vesicular removal by oligodendrocytes of membrane attack complexes formed by activated complement.</a> Nature. 339, 620-622 (1989).</li><li>Keyel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptolysin+O+clearance+through+sequestration+into+blebs+that+bud+passively+from+the+plasma+membrane.">Streptolysin O clearance through sequestration into blebs that bud passively from the plasma membrane.</a> J. Cell. Sci. 124, 2414-2423 (2011).</li><li>MacKenzie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+secretion+of+interleukin-1beta+by+microvesicle+shedding.">Rapid secretion of interleukin-1beta by microvesicle shedding.</a> Immunity. 15, 825-835 (2001).</li><li>Qu, Y., Franchi, L., Nunez, G., Dubyak, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical+IL-1+beta+secretion+stimulated+by+P2X7+receptors+is+dependent+on+inflammasome+activation+and+correlated+with+exosome+release+in+murine+macrophages.">Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages.</a> J. Immunol. 179, 1913-1925 (2007).</li><li>Andrei, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipases+C+and+A2+control+lysosome-mediated+IL-1+beta+secretion:+Implications+for+inflammatory+processes.">Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 9745-9750 (2004).</li><li>Franchi, L., Eigenbrod, T., Munoz-Planillo, R., Nunez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+caspase-1-activation+platform+that+regulates+immune+responses+and+disease+pathogenesis.">The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.</a> Nat. Immunol. 10, 241-247 (2009).</li><li>Magassa, N., Chandrasekaran, S., Caparon, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pyogenes+cytolysin-mediated+translocation+does+not+require+pore+formation+by+streptolysin+O.">Streptococcus pyogenes cytolysin-mediated translocation does not require pore formation by streptolysin O.</a> EMBO Rep. 11, 400-405 (2010).</li><li>Pinkney, M., Beachey, E., Kehoe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thiol-activated+toxin+streptolysin+O+does+not+require+a+thiol+group+for+cytolytic+activity.">The thiol-activated toxin streptolysin O does not require a thiol group for cytolytic activity.</a> Infect. Immun. 57, 2553-2558 (1989).</li><li>Watkins, S. C., Salter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+connectivity+between+immune+cells+mediated+by+tunneling+nanotubules.">Functional connectivity between immune cells mediated by tunneling nanotubules.</a> Immunity. 23, 309-318 (2005).</li><li>McNeil, P. L., Vogel, S. S., Miyake, K., Terasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patching+plasma+membrane+disruptions+with+cytoplasmic+membrane.">Patching plasma membrane disruptions with cytoplasmic membrane.</a> J. Cell. Sci. 113, 1891-1902 (2000).</li><li>Shannon, J. G., Ross, C. L., Koehler, T. M., Rest, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+anthrolysin+O,+the+Bacillus+anthracis+cholesterol-dependent+cytolysin.">Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.</a> Infect. Immun. 71, 3183-3189 (2003).</li></ol>

No conflicts of interest declared.

The techniques described here allow the examination of the responses of immune cells to bacterial toxins. The most critical step is the handling and dosing of the toxin. Toxin activity can be extremely variable, even between different aliquots of the same preparation, due to its fragility. This necessitates either testing each aliquot of toxin against a reference cell line or RBCs or using toxin gradients. Toxin gradients, as delivered by micropipette, allow the full spectrum of toxin-induced activities to be observed in...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Ni-NTA agarose</td>
 <td>Qiagen</td>
 <td>30210</td>
 <td></td>
 </tr>
 <tr>
 <td>polymixin-agarose</td>
 <td>Sigma</td>
 <td>P1411-5ML</td>
 <td></td>
 </tr>
 <tr>
 <td>Zeba Desalt Spin col</td>
 <td>Fisher</td>
 <td>PI-89891</td>
 <td></td>
 </tr>
 <tr>
 <td>sheep RBCs</td>
 <td>Fisher</td>
 <td>50-415-688</td>
 <td></td>
 </tr>
 <tr>
 <td>pBADgIII-SLO</td>
 <td>N/A</td>
 <td>N/A</td>
 <td>see ref9 </td>
 </tr>
 <tr>
 <td>Cy5 monoreactive dye</td>
 <td>GE Healthcare</td>
 <td>PA25001</td>
 <td></td>
 </tr>
 <tr>
 <td>Fura2-AM</td>
 <td>Life Technologies</td>
 <td>F1221</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcein AM/ Ethidium homodimer</td>
 <td>Life Technologies</td>
 <td>L3224</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-CD11c-APC</td>
 <td>BD Biosciences</td>
 <td>550261</td>
 <td></td>
 </tr>
 <tr>
 <td>collagen-coated glass-bottom dish</td>
 <td>Mattek</td>
 <td>P35GCol-1.5-10-C</td>
 <td></td>
 </tr>
 <tr>
 <td>femto-tip II</td>
 <td>Fisher</td>
 <td>E5242957000</td>
 <td></td>
 </tr>
 <tr>
 <td>Microloader</td>
 <td>Fisher</td>
 <td>E5242956003</td>
 <td></td>
 </tr>
 <tr>
 <td>dextran Alexa 555</td>
 <td>Life Technologies</td>
 <td>D34679</td>
 <td></td>
 </tr>
 <tr>
 <td>Injectman NI 2</td>
 <td>Eppendorf</td>
 <td>920000029</td>
 <td></td>
 </tr>
 <tr>
 <td>FemtoJet</td>
 <td>Eppendorf</td>
 <td>5247 000.013</td>
 <td></td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 1. List and source of specific reagents and equipment needed. Specific equipment and reagents used in this protocol, along with company and catalogue number are listed.</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td>Buffer</td>
 <td>Composition</td>
 <td>Step Used</td>
 </tr>
 <tr>
 <td>Lyse/Wash</td>
 <td>50 mM NaH2PO4 300 mM NaCl 10 mM imidazole, pH 8.0</td>
 <td>1.4</td>
 </tr>
 <tr>
 <td>Tris/salt</td>
 <td>50 mM Tris, pH 8.0 300 mM NaCl</td>
 <td>1.7</td>
 </tr>
 <tr>
 <td>Elution buffer</td>
 <td>50 mM Tris, pH 8.0 300 mM NaCl 250 mM imidazole</td>
 <td>1.8</td>
 </tr>
 <tr>
 <td>RBC Assay buffer</td>
 <td>0.3% BSA 2 mM CaCl2 10 mM HEPES, pH 7.4</td>
 <td>2.1</td>
 </tr>
 <tr>
 <td>buffer R/B</td>
 <td>RPMI cell culture medium 2 mM CaCl2 0.5% BSA</td>
 <td>3.1</td>
 </tr>
 </tbody>
</table>
</td>
 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>Table 2. List of buffers used in this protocol. The buffers used, their composition and the first step at which they are used in the protocol are listed.</td>
 </tr>
 </tbody>
</table>

visualization of bacterial toxin induced responses using live cell fluorescence microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Typically 107-108 U/ml SLO can be obtained with a protein concentration of 4 mg/ml. The amount of toxin required for cell lysis varies by cell type, but is usually 125-500 U/ml SLO (Figure 2B). Cell types like macrophages can be more resistant (4000 U/ml) though others (especially T cell lines) are more sensitive. These sensitivities correspond with commercially available SLO. Toxin activity decreases roughly 2-fold with each freeze-thaw, so hemolytic assays with each batch or thaw ...

Watch this Scientific Journal Video about Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy at JoVE.com

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

visualization-bacterial-toxin-induced-responses-using-live-cell

Research

JoVE Journal

Immunology and Infection

11.7K Views.  University of Pittsburgh School of Medicine. Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

Video: Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Application of Long-term cultured Interferon-&amp;#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.